Caspofungin at sub-inhibitory concentration promotes the formation of Candida albicans persister cells.

AIMS: Low caspofungin exposure is frequently encountered in patients with invasive candidiasis caused by Candida albicans. This study aimed to investigate the effects of caspofungin on C. albicans at sub-inhibitory concentrations. METHODS AND RESULTS: First, a comparative transcriptomics analysis was performed on C. albicans receiving caspofungin at sub-minimum inhibitory concentrations (sub-MICs). The results showed that caspofungin significantly changed the mRNA expression profile in DAY185, with DE-mRNAs enriched in the functions of cell wall biosynthesis, metabolism, etc. Subsequently, cellular fitness, cell aggregation, energy metabolism activity and the proportion of persister cells of C. albicans were quantitatively and/or qualitatively assessed after sub-MIC caspofungin exposure. No significant changes in cell fitness and aggregation formation were observed during treatment of C. albicans with sub-MIC caspofungin. In C. albicans aggregation treated with sub-MIC caspofungin, we observed a decrease in respiratory metabolism and an increase in persister cells; this effect was more pronounced in als1ΔΔ than in DAY185. CONCLUSIONS: Pre-exposure to sub-MIC caspofungin suppresses C. albicans respiratory metabolism and promotes persister cell development. SIGNIFICANCE AND IMPACT OF THE STUDY: Caspofungin should be used with caution in patients with C. albicans infections, as anti-infection therapy may fail due to persister cells.

Immunological characterization of the American cockroach allergen Per a 9 expressed in baculovirus-infected insect cells.

American cockroach (CR) allergy has been recognized as important IgE-mediated type I hypersensitivity. Per a 9 is an arginine kinase, reacting with IgE in sera of all CR allergic Thai patients. Per a 9 gene was cloned and expressed in eukaryotic systems (baculovirus-infected insect cells). The expressed Per a 9 was purified by Nickel column. The antigenicities were analyzed by ELISA, immunoblot analysis and basophile activation test. The results show that 13 out of 16 (81.3%) sera from American CR patients reacted to Per a 9, confirming that Per a 9 is a major allergen of CR. The IgE reactivity of Per a 9 in the sera from American CR patients was increased 8.3-fold in comparison with the sera from healthy controls. Per a 9 at 1.0 µg/ml induced an approximately up to 5.6-fold increase in CD63 and CCR3 double positive cells when incubating with passively sensitized basophils from by sera from American CR patients.

Influence of Genetic Polymorphisms on Mycophenolic Acid Pharmacokinetics and Patient Outcomes in Renal Transplantation.

BACKGROUND: Mycophenolic Acid (MPA) is an immunosuppressive drug widely used in the treatment of organ transplantation and autoimmune diseases. Pharmacokinetics and pharmacodynamics of MPA varies between individuals, the potential reasons being the genetic polymorphisms in key enzymes, drug transporters and target proteins of MPA. OBJECTIVE: We try to provide pharmacogenomics information for drug selection and dose adjustment, aiming to improve drug efficacy and reduce side effects in clinical application of MPA. METHODS: In this review, we summarize the literatures in Pubmed that reported MPA-related Single Nucleotide Polymorphisms (SNPs) of renal transplant patients in recent 15 years. RESULTS: Genetic polymorphisms involving uridine diphosphate glucuronosyltransferase enzymes, organic anion transport polypeptides, multidrug resistance-associated protein 2, inosine monophosphate dehydrogenase and immune- response mediators may be associated with the metabolism, efficacy and toxicity of MPA, thus resulting in different MPA exposure and patient outcomes in renal transplantation. CONCLUSION: Several SNPs show significant association with MPA pharmacokinetics and pharmacodynamics, but conflicting results are reported, and no studies on MPA genetic polymorphisms have been translated into clinical practice. More prospective studies are needed to clear the role of genetic polymorphisms on MPA in renal transplantation patients.

Evolution of the protease-activated receptor family in vertebrates.

Belonging to the G protein-coupled receptor (GPcr) family, the protease-activated receptors (Pars) consist of 4 members, PAR1-4. PARs mediate the activation of cells via thrombin, serine and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis and other normal pathological processes. In the present study, we examined the evolution of PARs by analyzing phylogenetic trees, chromosome location, selective pressure and functional divergence based on the 169 functional gene alignment sequences from 57 vertebrate gene sequences. We found that the 4 Pars originated from 4 invertebrate ancestors by phylogenetic trees analysis. The selective pressure results revealed that only PAR1 appeared by positive selection during its evolution, while the other PAR members did not. In addition, we noticed that although these PARs evolved separately, the results of functional divergence indicated that their evolutional rates were similar and their functions did not significantly diverge. The findings of our study provide valuable insight into the evolutionary history of the vertebrate PAR family.

Integrative genomic analyses of the RNA-binding protein, RNPC1, and its potential role in cancer prediction.

The RNA binding motif protein 38 (RBM38, also known as RNPC1) plays a pivotal role in regulating a wide range of biological processes, from cell proliferation and cell cycle arrest to cell myogenic differentiation. It was originally recognized as an oncogene, and was frequently found to be amplified in prostate, ovarian and colorectal cancer, chronic lymphocytic leukemia, colon carcinoma, esophageal cancer, dog lymphomas and breast cancer. In the present study, the complete RNPC1 gene was identified in a number of vertebrate genomes, suggesting that RNPC1 exists in all types of vertebrates, including fish, amphibians, birds and mammals. In the different genomes, the gene had a similar 4 exon/3 intron organization, and all the genetic loci were syntenically conserved. The phylogenetic tree demonstrated that the RNPC1 gene from the mammalian, bird, reptile and teleost lineage formed a species-specific cluster. A total of 34 functionally relevant single nucleotide polymorphisms (SNPs), including 14 SNPs causing missense mutations, 8 exonic splicing enhancer SNPs and 12 SNPs causing nonsense mutations, were identified in the human RNPC1 gene. RNPC1 was found to be expressed in bladder, blood, brain, breast, colorectal, eye, head and neck, lung, ovarian, skin and soft tissue cancer. In 14 of the 94 tests, an association between RNPC1 gene expression and cancer prognosis was observed. We found that the association between the expression of RNPC1 and prognosis varied in different types of cancer, and even in the same type of cancer from the different databases used. This suggests that the function of RNPC1 in these tumors may be multidimensional. The sex determining region Y (SRY)-box 5 (Sox5), runt-related transcription factor 3 (RUNX3), CCAAT displacement protein 1 (CUTL1), v-rel avian reticuloendotheliosis viral oncogene homolog (Rel)A, peroxisome proliferator-activated receptor γ isoform 2 (PPARγ2) and activating transcription factor 6 (ATF6) regulatory transcription factor binding sites were identified in the upstream (promoter) region of the RNPC1 gene, and may thus be involved in the effects of RNPC1 in tumors.

In silico prediction of the T-cell and IgE-binding epitopes of Per a 6 and Bla g 6 allergens in cockroaches.

Per a 6 and Bla g 6 are cockroach allergens found in Periplaneta americana and Blattella germanica, respectively. The objective of the present study was to predict the B­ and T­cell epitopes of the Per a 6 and Bla g 6 allergens. Three immunoinformatics tools, the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server, were used to predict the potential B­cell epitopes, while Net­MHCIIpan­2.0 and NetMHCII­2.2 were used to predict the T­cell epitopes of the two allergens. As a result, seven peptides were predicted in the Per a 6 allergen and seven peptides were predicted in the Bla g 6 allergen in the B­cell epitope predictions. In the T­cell prediction, the Per a 6 allergen was predicted to have nine strongly binding nonamer core epitope sequences (IC50<50 nm) and 28 weakly binding sequences (50 nm

In Silico Prediction of T and B Cell Epitopes of Der f 25 in Dermatophagoides farinae.

The house dust mites are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases. Der f 25 is a triosephosphate isomerase, representing the major allergen identified in Dermatophagoides farinae. The objective of this study was to predict the B and T cell epitopes of Der f 25. In the present study, we analyzed the physiochemical properties, function motifs and domains, and structural-based detailed features of Der f 25 and predicted the B cell linear epitopes of Der f 25 by DNAStar protean system, BPAP, and BepiPred 1.0 server and the T cell epitopes by NetMHCIIpan-3.0 and NetMHCII-2.2. As a result, the sequence and structure analysis identified that Der f 25 belongs to the triosephosphate isomerase family and exhibited a triosephosphate isomerase pattern (PS001371). Eight B cell epitopes (11-18, 30-35, 71-77, 99-107, 132-138, 173-187, 193-197, and 211-224) and five T cell epitopes including 26-34, 38-54, 66-74, 142-151, and 239-247 were predicted in this study. These results can be used to benefit allergen immunotherapies and reduce the frequency of mite allergic reactions.

Cytotoxic polyphenols against breast tumor cell in Smilax china L.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax china L., referred to 'Ba Qia' (or 'Jin Gang Teng') in China, is a small vine that grows in the southern parts of China. The roots and tubers of S. china L. have been applied not only as traditional Chinese medicine (TCM) for treatment of diuretic, rheumatic arthritic, detoxication, lumbago, gout, tumor, and inflammatory diseases, but also as food in some area of China. AIM OF STUDY: To investigate the breast tumor cell toxic components in S. china L. continuously and systematically. MATERIALS AND METHODS: Three fractions and six polyphenols were isolated from roots and tubers of S. china L. under bioassay-guided screenings. The structures of six compounds were elucidated by spectroscopic methods and comparison with published data. Their breast tumor cytotoxicity and apoptosis of purified components were performed. RESULTS: Six polyphenols were obtained on the basis of a bioassay-guided separation of the ethyl acetate extract, and their breast tumor cytotoxic activities were tested. They showed anti-tumor activities against MCF-7 and MDA-MB-231 with IC50 value of 2.1-38.9 microg/mL, and can induce apoptosis for MCF-7 and MDA-MB-231. CONCLUSIONS: Among these six polyphenols, five (1, 3-6) were reported for the 1st time with in vitro activities on anti-breast tumor cell. It is likely that these polyphenols are the active components of S. china L. responsible for the anti-breast tumor cell activities.

Human tissue-specific microenvironment: an essential requirement for mouse models of breast cancer.

An ideal mouse model should closely mimic a clinical situation. However, for most models available, this is not the case since clinical trials frequently fail to reproduce the highly encouraging therapeutic results obtained from pre-clinical studies performed using mouse models. In this study, in the process of extending the application of our previously established breast tissue-derived orthotopic and metastatic (BOM) model, the human breast cancer cell line MDA-MB-231 failed to exhibit any osteotropic features that differed from previous studies. Our observations suggest that a human tissue-specific microenvironment could be an essential requirement for a successful mouse model of breast cancer. Here, multiple in vivo breast cancer models were used to confirm this hypothesis. Human breast tissue and cancellated bone were transplanted subcutaneously to female severe combined immunodeficiency disease (SCID) mice by different assemblies, to build several mouse models termed 'breast-breast', 'breast-bone', 'bone-bone', 'MFP (mouse mammary fat pad)-bone', and 'MFP-breast' models. Two human breast cancer cell lines, MDA-MB-231 and MDA-MB-231BO, and the mouse breast cancer cell line TM40D were used. All cancer cells were labeled with GFP for gross observation. In addition, transplanted human tissues and various mouse tissues including bone, lung, liver, mesentery were examined microscopically. Based on morphological, immunohistochemical, and enzymohistochemical evidence obtained from several comparative experiments in 'breast-breast', 'breast-bone' and 'bone-bone' models, the BOM model was proved to be feasible and reliable. The organ tropism of the breast cancer cell line, which was derived from a mouse model by intracardiac inoculation in a pure mouse microenvironment, was reconsidered. The behavior of breast cancer cells in the mouse model was altered in response to the varying microenvironment. The results in this study suggest the human tissue-specific micro-environment is most likely an essential requirement in mouse models of breast cancer.

Cytotoxic and antibacterial triterpenoids derivatives from Clematis ganpiniana.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and rhizomes of Clematis are commonly used as an analgesic, abirritative, antibacterial, antiphlogistic, anticancer and diuretic agent. The Naxi people traditionally used Clematis ganpiniana's (Lévl. Et Vant.) as a diuretic agent, an anti-inflammatory and anticancer remedy. AIM OF STUDY: To investigate the cytotoxic and antibacterial components from Clematis ganpiniana. MATERIALS AND METHODS: The aboveground part of Clematis ganpiniana was isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods and comparison with published data. Their cytotoxic, apoptosis and antibacterial activities of purified components were also performed. RESULTS: By bioassay-guided fractionation techniques and chemical characterization, four triterpene glycosides were isolated and their cytotoxicity against cancer cells and antibacterial activity were tested. They showed significant inhibitory activities against MCF-7, MDA-MB-231 with IC50 value of 0.7-16.5 microg/ml, and significant apoptosis for MCF-7 and MDA-MB-231. Moreover, compound 4 showed weak wide-spectrum antibacterial activity. CONCLUSION: These results provide promising baseline information for the potential use of Clematis ganpiniana as well as some of the isolated compounds in the treatment of cancer and infectious disease.

Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom.

L-amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin-Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55kDa in SDS-PAGE and an apparent molecular weight of 70kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against L-Tyr, L-Asp, L-Phe, L-Glu, L-Trp, L-His, L-Gln, L-Ile, L-Met, L-Leu and moderately active against L-Lys, L-Arg, L-Ala and L-Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite.

Filamentous-actins in human hepatocarcinoma cells with CLSM.

AIM: To establish a method for optical sections of HepG2 human hepatoblastoma cells with confocal laser scanning microscope (CLSM) and to study the spatial structure of filamentous actin (F-actin) in HepG2 cells. METHODS: HepG2 cells were stained with FITC-phalloidin that specifically binds F-actin, with propidium iodide (PI) to the nucleus, and scanned with a CLSM to generate optically sectioned images. A series of optical sections taken successively at different focal levels in steps of 0.7 microm were reconstructed with the CLSM reconstruction program. RESULTS: CLSM images showed that the FITC-stained F-actin was abundant microfilament bundles parallel or netted through the whole cell and its processes. Most F-actin microfilaments extended through the cell from one part toward the other or run through the process. Some microfilaments were attached to the plasma membrane, or formed a structural bridge connecting to the neighboring cells. CONCLUSION: A method for double labeling HepG2 human hepatoblastoma cells and CLSM imaging F-actin microfilaments and nuclei by image thin optical sections and spatial structure was developed. It provides a very useful way to study the spatial structure of F-actin.